Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Bacteriology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
STRUCTURAL BIOLOGY

S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus

Matthew J. Ford, John F. Nomellini, John Smit
Matthew J. Ford
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John F. Nomellini
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John Smit
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jsmit@interchange.ubc.ca
DOI: 10.1128/JB.01690-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first ∼225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first ∼225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.

Bacterial surface layers (S-layers) are composed of protein or glycoprotein subunits that assemble on the outermost surface of the cell, forming a two-dimensional lattice that covers the organism (5). Most S-layers are composed of a single protein or glycoprotein species, with molecular masses ranging from 40 to 200 kDa (30). Despite a large number of S-layer-possessing organisms and the substantial knowledge accumulated regarding S-layer structure, assembly, biochemistry, and genetics (32), relatively little is known about attachment of the S-layer to the cell surface, especially in the case of gram-negative bacteria.

S-layers from gram-positive bacteria remain surface associated by binding to carbohydrate moieties of the peptidoglycan and/or lipoglycans in the cell wall (30). S-layers bind to these carbohydrates via an amino acid motif in the S-layer protein, the so-called surface layer homology (SLH) domain (24). The SLH domain is a conserved sequence of approximately 55 amino acids, usually repeated in tandem three times (24).

In contrast, no general S-layer anchoring motif has been identified in gram-negative bacteria. Available evidence in each case so far implicates the involvement of the N terminus of S-layer protein monomers. The S-layer of Aeromonas salmonicida may be cell surface anchored via the N termini of the S-layer subunits since they are inaccessible to trypsin (13). Similarly, the Campylobacter fetus S-layer is cell surface anchored via the N terminus of the C. fetus S-layer monomer, SapA, since N-terminal SapA deletions disrupt SapA anchoring (14) while C-terminal SapA truncations do not (43).

The S-layer of the gram-negative bacterium Caulobacter crescentus follows this general pattern; the N terminus of the S-layer protein (RsaA) is thought to mediate direct anchoring of the S-layer to the C. crescentus cell surface. Mutations (8) and truncations (9) in the RsaA extreme N terminus lead to an S-layer shedding phenotype. A species of smooth lipopolysaccharide (SLPS), whose O side chain is composed at least in part of N-acetylperosamine, is found on the outer membrane of C. crescentus (2). This SLPS is at least involved in S-layer anchoring, since strains deficient in O side chain biogenesis shed S-layer (2), (40). Much of the LPS is not derivatized with O side chain and has no role in attachment. Thus far, we have not been able to accurately determine what fraction of LPS is modified by O side chain addition.

RsaA is a 98-kDa protein (1,025 amino acids) (19) that is secreted to the cell surface by an ABC transporter (type I secretion) (1). The S-layer proteins of C. fetus (35) and Serratia marcescens (23) are also secreted by a type I apparatus. However, most S-layer secretion in gram-negative bacteria occurs by type II secretion, a terminal branch of the general secretory pathway (11). In contrast to type II, type I secretion exports substrates to the cell surface without exposure to the periplasm and utilizes a noncleaved C-terminal secretion signal. Two well-described type I secretion systems are those required for the secretion of Escherichia coli hemolysin, HlyA (6), and a Pseudomonas aeruginosa alkaline protease, AprA (28).

After secretion, RsaA assembles into a two-dimensional hexagonal lattice on the cell surface (34). Some RsaA monomers are bound to the cell surface by interacting with SLPS (2), while we presume others remain surface associated by interacting with attached RsaA monomers via the subunit-subunit interactions that result in the hexagonal array. Assembly into a hexagonal array requires calcium (34, 41), which may mediate RsaA crystallization via calcium bridging between RsaA monomers (34). A calcium-binding motif located near the C terminus of RsaA, the so-called RTX (repeats in toxin) motif (19), likely mediates this process. A characteristic of type I secreted proteins, the RTX motif is a sequence of nine amino acids [L-X-G-G-X-G-(N/D)-D-X] repeated in tandem (reviewed in reference 42). Perturbations in RsaA at or near the RTX repeats results in the shedding of RsaA (7, 8), presumably by affecting calcium-mediated RsaA subunit-subunit interactions that would otherwise result in indirect tethering of many monomers to the cell surface.

Indeed, it is the complication that perturbation of crystallization leads to the same gross phenotype (shedding) as loss of direct SLPS attachment, coupled with our lack of knowledge about how much SLPS is present on the bacterium, that has made it difficult to precisely address the specifics of attachment. For example, one possibility in addition to N-terminal attachment is that a secondary attachment site resides elsewhere on RsaA. Alternatively, it may be that crystallization leads to the proper folding that is required even for N-terminal attachment. For this reason we developed methods in this report to dissect attachment from the crystallization function and show that SLPS is required for C. crescentus RsaA anchoring. We also demonstrate that RsaA amino acids 1 to 277 are sufficient for RsaA anchoring. Surprisingly, all perturbations created within the first ∼225 RsaA residues cause loss of RsaA anchoring.

As a by-product of S-layer anchoring studies, we learned more about the localization of the C. crescentus S-layer-editing metalloprotease, Sap. Because Sap did not appear to contain (i) a signal leader peptide at the N-terminal, (ii) a type I C-terminal secretion signal, or (iii) obvious transmembrane domains, it was previously suggested that Sap is an intracellular enzyme. The current study challenges this notion, suggesting that Sap is an outer membrane-bound enzyme that is secreted to the cell surface primarily by the S-layer type I mechanism.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.All of the strains and plasmids used in this study are listed in Tables 1 and 2, respectively. E. coli DH5α was used for most E. coli cloning manipulations and was grown at 37°C in Luria broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract) with 1.3% agar for plates. C. crescentus strains were grown at 30°C in PYE (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) medium with 1.2% agar for plates. Ampicillin, kanamycin (KAN), and streptomycin were used at 50 μg/ml, and chloramphenicol (CHL) was used at 20 μg/ml for E. coli and 2 μg/ml for C. crescentus. KAN and streptomycin were used at 25 μg/ml when used along with CHL for C. crescentus.

View this table:
  • View inline
  • View popup
TABLE 1.

Bacterial strains used in this study

View this table:
  • View inline
  • View popup
TABLE 2.

Plasmids used in this study

Plasmid and DNA manipulations.Standard methods of DNA manipulation were used (29). Electroporation of C. crescentus was performed as previously described (20). PCR products were generated using Platinum Pfx DNA polymerase (Invitrogen). Sequences of the primers used in this study are detailed in Table 3. Plasmids to be digested with ClaI were produced from RB404, a non-DNA methylating strain of E. coli.

View this table:
  • View inline
  • View popup
TABLE 3.

Primers used in this study

C. crescentus expression vectors. (i) p4A.p4A is a vector derived from pUC8 (25) that contains a full-length copy of rsaA under control of a modified rsaA promoter region. It contains an oriV-derived plasmid RSF1010 (39), which allows for replication in E. coli and in C. crescentus strains that express repBAC. Oligonucleotides 1060 and 1920 were used for inverse PCR of pUC8. The PCR product was digested with StuI and ScaI and ligated to the HpaI-digested Cm resistance gene called CHE, resulting in pUC8CX. CHE was made using the oligonucleotides JNCHE-1 and JNCHE-2 to PCR amplify the gene from pMMB206 (26). The oriV was inserted by digesting pUC8CX with EcoO109, filling in the resulting recessed ends with Klenow polymerase, and then digesting with HindIII; this small fragment was replaced with the 521-bp HindIII/XmnI oriV fragment from pCR2.1oriV (39), resulting in pUC8CVX. The lac promoter was replaced with a modified rsaA promoter region as follows: the EcoRI/HindIII fragment from pSSa49ΔSD (10) containing a modified rsaA promoter region was ligated into pUC8, resulting in pUC8ΔSD. pUC8ΔSD and pUC8CVX were digested with EcoRI and ligated. This plasmid was digested with SapI and PstI, filled in with Klenow polymerase, ligated, and selected on CHL. This last manipulation removed all of pUC8ΔSD except the modified rsaA region, now upstream of the EcoRI/HindIII multiple cloning site region. This plasmid, pUC8CVXΔSD, was renamed p4A for simplicity. All full-length rsaA constructs can be cloned into this high-copy-number vector as EcoRI/HindIII fragments and transcribed from the modified rsaA promoter region. Wild-type (WT) rsaA from pTZ18UB:rsaAΔP (8) in p4A is p4A-WT.

(ii) pBBR3ΔSD. pBBR3ΔSD was used as a E. coli and C. crescentus shuttle vector and does not require repBAC to replicate. To create this vector, p4A-WT was digested with NspI, and the recessed ends were filled in with Klenow polymerase. The linearized plasmid was ligated into pBBR3 (36) digested with SmaI, creating a 12-kb intermediate plasmid. The plasmid was digested with HindIII, releasing most of p4A-WT, leaving the modified rsaA promoter region with pBBR3 vector. The remaining fragment was circularized by ligation, resulting in pBBR3ΔSD.

Construction of plasmids carrying rsaA with collagenase cleavage sites. (i) pUC9CXSMCC.pUC9CXSMCC was created by annealing oligonucleotides MCC-1 and MCC-2 and ligating them into XhoI-StuI-digested pUC9CXS (7) in the same orientation as the promotorless Cm resistance gene. These oligonucleotides encode, from 5′ to 3′, a factor X protease cleavage site, followed by two collagenase cleavage sites in tandem (PHGPAGP).

(ii) pWB9:rsaAΔP:Hps1MCCΔ, pWB9:rsaAΔP:Hps4MCCΔ, and pWB9:rsaAΔP:Hps12MCCΔ.pUC9CXSMCC was digested with BamHI, releasing a fragment that included the MCC and a promotorless Cm resistance gene. The BamHI cassette was then ligated into the unique BamHI site in either pTZ18UB:rsaA(HinPI277BamHI) (7), pTZ18UB:rsaA(HinPI690BamHI) (7), or pTZ18UB:rsaA(HinPI723BamHI) (7) to place the MCC cassette at the position in rsaA corresponding to amino acid 277, 690, or 723, respectively. Proper orientation of the MCC cassette was achieved by selecting for Cm resistance (the Cm resistance gene is driven by the lac promoter in pTZ18). The Cm resistance gene was then excised by digestion with BglII, and plasmids were recircularized by ligation, creating intermediate plasmids. The EcoRI-SstI fragments of these plasmids were excised and ligated into pWB9, resulting in pWB9:rsaAΔP:Hps1MCCΔ, pWB9:rsaAΔP:Hps4MCCΔ, and pWB9:rsaAΔP:Hps12MCCΔ.

(iii) pWB9:rsaAΔP:B162MCCΔ.pBSKIIφBamHI was constructed by cutting pBSKII with BamHI and filling in with Klenow polymerase, and the product was religated, thereby destroying the BamHI site. Next, the rsaA EcoRI/ClaI fragment was excised from pTZ18UB:rsaA(HinPI723BamHI) (7) and ligated into pBSKIIφBamHI. Mutation at RsaA amino acids 162/163 was achieved using Quickchange (Stratagene, La Jolla, CA), using the primers B162F and B162R, changing RsaA amino acids F162/L163 to G162/S163 and creating a BamHI site. The MCC cassette from pUC9CXSMCC was inserted into this BamHI site and the Cm resistance gene was removed as described above, yielding pBSKIIφBamHI:rsaAEcoRI-ClaIBamHI162MCCΔ. p4A-WT was digested with PstI, releasing a PstI-PstI fragment containing two ClaI sites from a noncoding region of the plasmid. The remaining backbone plasmid was recircularized by ligation, creating p4A-WTΔPstI. Next, the rsaA EcoRI/ClaI fragment with the desired mutation, excised from pBSKIIφBamHI:rsaAEcoRI-ClaIBamHI162MCCΔ, was inserted by ligation into p4A-WTΔPstI. To replace the PstI-PstI fragment in this intermediate plasmid (which contained an SstI site required for the next step), the AvrII/HindIII fragment (containing the PstI-PstI fragment), obtained from digesting p4A-WT with AvrII and HindIII, was ligated into the intermediate plasmid. Finally, the mutant rsaA-encoding EcoRI/SstI fragment was ligated into pWB9 as described above, yielding pWB9:rsaAΔP:B162MCCΔ.

Construction of plasmids carrying rsaA with collagenase cleavage at RsaA position 277 with additional mutations in the RsaA N terminus. (i) pWB9:rsaAΔP:B7Hps1MCCΔ, pWB9:rsaAΔP:B154Hps1MCCΔ, and pWB9:rsaAΔP:B222Hps1MCCΔ.pTZ18UB:rsaA(HinPI277BamHI)MCCΔ was used as a template for PCR for site-directed mutagenesis of rsaA to achieve “BamHI” mutations at RsaA positions 7, 154, and 222 using Quickchange (Stratagene). Primers B7F and B7R were used to change RsaA Q7/L8 to G7/S8, primers B154F and B154R were used to change V154/D155 to G154/S155, and primers B222F and B222R were used to change D222/L223 to G222/S223. The EcoRI-SstI fragments from the Quikchange products were excised and ligated into pWB9 as described above, yielding pWB9:rsaAΔP:B7Hps1MCCΔ, pWB9:rsaAΔP:B154Hps1MCCΔ, and pWB9:rsaAΔP:B222Hps1MCCΔ.

(ii) pWB9:rsaAΔP:Taq29Hps1MCCΔ, pWB9:rsaAΔP:Mps4Hps1MCCΔ.The EcoRI-NotI fragments from pTZ18UB:rsaA(TaqI29BamHI) (8) or pTZ18UB:rsaA(MspI69BamHI) (7) were ligated into pTZ18UB:rsaA(HinPI277BamHI)MCC. The rsaA EcoRI/SstI fragments were cloned into pWB9 as described above, yielding pWB9:rsaAΔP:Taq29Hps1MCCΔ and pWB9:rsaAΔP:Mps4Hps1MCCΔ.

(iii) pBBR3ΔSD:Taq169Hps1MCCΔ.pTZ18UB:rsaA (HinPI277BamHI)MCCΔΔPstI was constructed as described above to remove two ClaI sites in the noncoding PstI-PstI fragment. The rsaA EcoRI/ClaI fragment from pTZ18UB:rsaA(TaqI169BamHI) (8) was ligated into pTZ18UB:rsaA (HinPI277BamHI)MCCΔΔPstI. From the resulting plasmid, the rsaA EcoRI/HindIII fragment was ligated into pBBR3ΔSD, yielding pBBR3ΔSD:Taq169Hps1MCCΔ.

Plasmids used for gene disruptions. (i) pK18mobsacBmanBΔNΔC.pK18mobsacBmanBΔNΔC was used to create a strain that was null for ManB and, consequently, SLPS. A PCR product encoding manB that was deleted in the regions encoding the N and C termini of ManB was generated using NA1000 chromosomal DNA and the primers ManB 169 and IManB 1202 and then blunt-end ligated into EcoRV-cut pBSKIIEEH (37). The EcoRI/HindIII fragment from the resulting plasmid was then ligated into pK18mobsacB (31), yielding pK18mobsacBmanBΔNΔC.

(ii) pK18mobsacB:pwbΔR.pK18mobsacB:pwbΔR was used to create an internal deletion in the S-layer-associated protease gene, sap. A PCR product encoding sap was created using JS4000 chromosomal DNA as the template and primers PWB_F and PWB_R and blunt-end ligated into EcoRV-digested pBSKII. sap was excised from the resulting plasmid as an EcoRI/HindIII fragment and ligated into pBSKIIESH (37). The resulting plasmid was digested with PstI, which removed 1,023 bp of the sap sequence (approximately one-half of the sap gene), and the remaining plasmid was recircularized by ligation. The internally deleted form of sap was excised as an EcoRI/HindIII fragment and ligated into pK18mobsacB, yielding pK18mobsacB:pwbΔR.

(iii) pK18mobsacB:rsaF aΔKP.pK18mobsacB:rsaF aΔKP was used for the internal deletion of rsaF a. A PCR product containing rsaF a and flanking regions of 1,008 bp 5′ and 139 bp 3′ was generated using NA1000 chromosomal DNA as a template and primers rsaF aΔKP_F and rsaF aΔKP_R. This fragment was blunt-end ligated into the pBSKI+ plasmid at the EcoRV site, resulting in pBSKI:rsaF aEX. pBSKI:rsaF aEX was digested with KpnI and PstI, blunt ended using T4 DNA polymerase, and recircularized by ligation. The resulting plasmid had an 852-bp internal deletion. This deleted version of rsaF a was excised as an EcoRI-HindIII fragment and ligated into pK18mobsacB, yielding pK18mobsacB:rsaF aΔKP.

(iv) pK18mobsacBrsaA353ΦB.pK18mobsacBrsaA353ΦB was used to disrupt the rsaA gene. The BamHI site in pTZ18UB:rsaA(AciI353BamHI) (7) was destroyed by digesting with BamHI and filling in with Klenow polymerase, and then the product was recircularized by ligation. This rendered the rsaA sequence out of frame and introduced an amber codon at a position corresponding to RsaA amino acid 358. The EcoRI/HindIII fragment containing the mutated rsaA gene was then ligated into pK18mobsacB, yielding pK18mobsacBrsaA353ΦB.

Construction of gene disruptions. (i) JS1010.Knockout of rsaF a in NA1000 was done using pK18mobsacB:rsaF aΔKP. Primary recombination of the plasmid was selected using Km resistance. Secondary selection on 5% sucrose PYE plates and subsequent replica plating on PYE and PYE KAN plates were used to confirm a second recombination event. rsaF a deletion was confirmed by PCR using primers JS1010_F and JS1010_R.

(ii) JS1011.Knockout of rsaF a in JS1008 (36) was done as described above for the knockout of rsaF a in NA1000, except that colonies were screened by colony Western blot analysis (7) using RsaA antiserum. Colonies that were S-layer negative according to the colony Western blot analysis were confirmed by PCR. A strain possessing the internally deleted rsaF a and CHL cassette-interrupted rsaF b was then subjected to rsaA deletion, using pAL1. The deletion was confirmed by PCR using primers JS1011_F and JS1011_R.

(iii) JS1012.pK18mobsacB:pwbΔR was used to knockout the sap gene in JS1001 (15), replacing the wild-type gene with an internally deleted version of sap missing about one-half of the gene sequence. Recombinants were selected as described for JS1010. Gene replacement was confirmed by PCR using primers PWB_F and PWB_R. The rsaA gene was deleted using pAL1. Recombinants were selected as described for JS1011. Colonies were screened by PCR using primers JS1011_F and JS1011_R.

(iv) JS1013.pK18mobsacBrsaA353ΦB was used to disrupt rsaA in NA1000. Recombinants were selected for as described for JS1010 and were screened by colony Western blot analysis (7) using RsaA antiserum. Loss of RsaA was confirmed by low-pH extraction (see below).

(v) JS1014.pK18mobsacBmanBΔNΔC was used to knock out manB in JS1013. Recombination of pK18mobsacBmanBΔNΔC was selected for using Km resistance.

(vi) JS4022.JS4022 was constructed by introduction of repBAC into recA of JS4015 in the same way that resulted in JS4019, as described previously (39).

(vii) JS4023.Knockout of rsaF a in JS4000 was done using pK18mobsacB:rsaF aΔKP as described for creation of JS1010.

(viii) JS4024.Knockout of manB in JS4015 (38) was achieved using pK18mobsacBmanBΔNΔC in the same manner used to create strain JS1014.

(ix) JS4025.Knockout of rsaF b in JS4023 was done via insertional inactivation using an N- and C-terminally deleted rsaF b fragment. The nonreplicating pTZ18UCHE:rsaF bΔNΔC (37) was electroporated into JS4023, selecting for Cm resistance. rsaF b insertion was confirmed by PCR using primers 1060 and JS4025_R.

S-layer reattachment assays. (i) Coculturing assay.Coculturing of S-layer donors (JS1001) with S-layer recipients (JS1003 and JS1004) was done as follows. Strains JS1001, JS1003, and JS1004 were grown to mid-log phase. An aliquot of JS1001 and either JS1003 or JS1004 in a cellular ratio of eight donors:one recipient was used to inoculate 10 ml of PYE medium. These cocultures were grown to mid-log phase and filtered through Whatman no. 52 hardened filter paper (Kent, England) to remove any aggregated protein. Equivalent numbers of cells (assessed by the optical density at 600 nm [OD600]) were then subjected to low-pH extraction (see “Protein techniques” below) to assess the extent of S-layer reattachment.

(ii) RsaA reattachment assay.To produce soluble protein for reattachment purposes, 10-ml cultures of SLPS-negative cultures (JS1001, for wild-type RsaA) or of SLPS-negative, S-layer-negative C. crescentus strains that are Sap positive (JS1004) or Sap negative (JS1012) harboring the plasmids encoding RsaA or RsaA mutants were grown to an OD600 of ∼0.9 and pelleted by centrifugation three times at 13,000 ×g for 10 min at 4°C; the pellet was discarded after each centrifugation in an effort to clear the supernatant of cells. S-layer-negative target cells possessing various levels of SLPS (JS1003, JS1004, and JS4024) were grown to an OD600 of ∼0.9 and then pelleted by centrifugation at 13,000 × g for 5 min at 4°C. Cell pellets were washed with 1 ml of PYE medium, subjected to centrifugation, and then suspended with the supernatants containing the soluble RsaA or RsaA mutant. The volume of supernatant used for resuspension was 1.2 to 1.5 times the volume of the target cells initially pelleted. The resulting mixtures were incubated at room temperature for 10 to 30 min with slow inversion. These cultures were then pelleted, washed twice with 10 mM HEPES (pH 7) for subsequent low-pH extraction or washed twice with 10 mM Tris-HCl (pH 8) for subsequent boiling of the entire sample or for subsequent whole-cell protein preparations.

Protein techniques. (i) Low-pH extraction.Surface protein from C. crescentus cells was extracted by low-pH extraction or by EGTA treatment as previously described (41). To compare the amounts of S-layer protein from C. crescentus cells expressing RsaA to S-layer that had been reattached to S-layer-negative cells, equivalent amounts of cells (determined by the OD600) growing at log phase were used, and equal amounts of extracted protein samples were loaded onto protein gels.

(ii) Whole-cell protein preparations.Whole-cell protein preparations were done using equivalent amounts of cells (determined by the OD600) taken from 10-ml C. crescentus cultures growing at log phase. Cultures were centrifuged, and the cell pellets were washed twice with 10 mM Tris-HCl, pH 8. The cells were resuspended in 100 μl of 10 mM Tris-HCl, pH 8, and lysozyme (100 μg/ml) was added; the mixture was then incubated at 25°C for 15 min. RNase A (50 μg/ml) and DNase I (1 μg/ml) were added, and incubation continued at 37°C for 30 min. Equal volumes of whole-cell protein preparations were loaded onto protein gels. Some samples were subjected to low-pH extraction before whole-cell protein preparation was done to disrupt RsaA monomer-monomer interactions in order to assess RsaA that was directly anchored to the cell surface. As a rapid alternative to this method, for some experiments equivalent numbers of cells were washed twice with 10 mM Tris-HCl, pH 8, and boiled for 5 min.

(iii) Collagenase digests.Collagenase digests performed on soluble RsaA carrying a collagenase recognition sequence were done in PYE medium supplemented with collagenase buffer as follows. Supernatant from S-layer-shedding C. crescentus strains were harvested as described above (see “RsaA reattachment assay”). The resulting culture supernatant was supplemented to achieve final concentrations of 10 mM NaCl, 4 mM CaCl2, 2 mM Tris-HCl (pH 7), and 2 mM β-mercaptoethanol. Type III collagenase (Sigma-Aldrich, St. Louis, MO) was added to 7 U/ml, and digestion was performed for various lengths of time at 37°C.

(iv) SDS-PAGE and Western blotting.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) used 7.5%, 10%, or 12% separating gels as appropriate for the anticipated protein sizes. Coomassie-stained SDS-PAGE gels and Western immunoblotting were done by standard methods (29); for Western analysis horseradish peroxidase-coupled secondary antibodies were used for colorimetric detection. Antiserum of RsaA with a deletion of residues 188 to 784(37) was used at a 1/10,000 dilution, SLPS antiserum (40) was used at a 1/6,000 dilution, and RsaFa antiserum (22), which was raised against RsaFa but is also reactive with RsaFb, was used at a 1/1,000.

RESULTS

Comparison of RsaA preparation methods for cell surface reassembly.One goal in assay development was to find a method of RsaA preparation that allowed for optimum levels of RsaA reassembly onto S-layer-negative cells. Full-length RsaA can be obtained by treating cells to low-pH conditions or EGTA extraction (41). Alternatively, SLPS-deficient strains shed RsaA into the culture medium, and much of this protein forms aggregates (2) that can be collected by filtration and solubilized by urea treatment. Previously established methods of RsaA preparation for subsequent reassembly onto lipid vesicles involved low pH and EGTA treatment of cells to release RsaA (27). Therefore, low-pH- and EGTA-extracted RsaA preparations were incubated with S-layer-negative cells that possessed either wild-type (JS1003) or deficient (JS1004) levels of SLPS in the normal C. crescentus growth medium PYE. RsaA obtained from both extraction methods reattached to cells in an SLPS-dependent manner (except for the EGTA-extracted RsaA that was not dialyzed against water prior to addition to S-layer-negative cells, probably due to a shortage of available Ca2+) (Fig. 1a). Slightly more low-pH-extracted protein appeared to reassemble in comparison to EGTA-extracted protein; however, neither approach led to S-layer levels comparable to those found on wild-type cells (Fig. 1a). Similar results were obtained when cells and protein were incubated in calcium-supplemented water rather than PYE medium (not shown).

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Anti-RsaA immunoblot analysis of S-layer reattachment onto C. crescentus cells. (a) S-layer reattachment. Wild-type RsaA was extracted from the C. crescentus cell surface by low-pH or EGTA treatment, and the resulting protein was dialyzed (dial) or not against water. These RsaA preparations were subsequently incubated with corresponding amounts of S-layer-negative cells that possess wild-type (JS1003; wt) or deficient (JS1004; d) levels of SLPS. Reattached RsaA was subsequently extracted by low-pH treatment. For comparison, the lane labeled as control (ctl) is a low-pH extraction of an equivalent number of cells expressing wild-type levels of S-layer. N/A, not applicable. (b) S-layer reattachment by coculturing S-layer donor (D) and recipient (R) cells. S-layer shedding cells (JS1001; donors) were cocultured with S-layer-negative cells (recipients) that possess wild-type (JS1003; R+) or deficient (JS1004; R−) levels of SLPS. Equivalent numbers of wild-type (NA1000; wt) cells alone, donor cells alone, donors plus wild-type SLPS recipients (D+R+), or donors plus deficient SLPS recipients (D+R−) were subjected to low-pH extraction. (c) S-layer reattachment using soluble RsaA from the supernatant of shedder cultures. Soluble RsaA harvested from the supernatant of an S-layer-shedding strain (JS1001) was incubated with a corresponding number of S-layer-negative cells that possess wild-type (wt) or deficient (d) levels of SLPS. Reattached RsaA was subsequently extracted by low-pH treatment. Lane 1 shows results of the 12-μl supernatant from the shedder strain. In lanes 2 and 3, to assess the extent of S-layer reattachment, low-pH extractions of 100% (lane 2) and 10% (lane 3) of the number of recipient cells used in the reattachment assay were performed on NA1000 cells. In lanes 4 to 7, results of low-pH extraction of S-layer-negative cells possessing wild-type levels of SLPS (JS1003; control, lane 4), deficient levels of SLPS (JS1004; control, lane 5), wild-type levels of SLPS (JS1003) after incubation with soluble RsaA (lane 6), and deficient levels of SLPS (JS1004) after incubation with soluble RsaA (lane 7) are shown.

Coculturing of an S-layer donor strain (with defective LPS that sheds S-layer protein) and an S-layer recipient strain (that possesses normal LPS but lacks the S-layer protein) achieves S-layer recrystallization in A. salmonicida (18). Accordingly, an S-layer-shedding strain (JS1001, donor) was cocultured with an S-layer-negative strain that possessed wild-type (JS1003, recipient) or deficient (JS1004, recipient) levels of SLPS. More S-layer reassembled selectively to SLPS-positive cells (JS1003) in this method compared to the previous methods (Fig. 1b). These results suggested that soluble, reassembly-competent RsaA was present in the supernatant of S-layer-shedding strains, in addition to the RsaA that forms aggregates. Accordingly, supernatant from an S-layer-shedding C. crescentus culture (JS1001) was analyzed, and soluble RsaA was indeed found in appreciable quantity (Fig. 1c, lane 1). RsaA obtained in this manner reassembled onto S-layer-negative (JS1003) cells at wild-type (NA1000) levels (Fig. 1c, compare lane 6 to lane 2).

Thus, the method of RsaA preparation strongly affected the ability of the protein to reassemble on the cell surface, and it was apparent that soluble secreted RsaA was the best source of RsaA variants to test regions or residues that might be important for RsaA anchoring.

SLPS is required for S-layer anchoring.Strains deficient in SLPS (such as JS1001) shed their S-layer into the culture medium. JS1001 was isolated as a strain able to grow in the absence of calcium which acquired an as yet undefined mutation that greatly reduced, but did not eliminate, SLPS production (2). In reattachment assays of RsaA to JS1004 (S-layer-negative version of JS1001), a low level of RsaA still reattached to the cells (Fig. 1c, lane 7, for example), maybe due to a low level of SLPS still present.

The N-acetylperosamine biosynthetic pathway was previously proposed (2), and disruption of manB, which encodes a putative phosphomannomutase involved in N-acetylperosamine synthesis, results in the loss of SLPS production (2). We disrupted manB in RsaA-negative strain JS1013, creating JS1014, a strain completely devoid of SLPS (Fig. 2a, lane 4). To determine if RsaA reattachment requires SLPS, soluble, reattachment-competent RsaA (isolated from the supernatant of JS1001) was incubated with S-layer-negative cells that had no SLPS (JS1014), were deficient in SLPS (JS1004), or had wild-type levels (JS1003). No RsaA reattachment was observed to JS1014, a small amount of RsaA reattached to JS1004, and a large amount of RsaA reattached to JS1003 (Fig. 2b), indicating that SLPS is required for S-layer anchoring.

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

SLPS and the attachment of S-layer. (a) SLPS levels in SLPS-positive, -deficient, and -negative C. crescentus strains. Immunoblot analysis using SLPS antiserum. Whole-cell protein preparations from equivalent numbers of cells were used. Lane 1, Km resistance cassette interruption of rsaA in spontaneous calcium-independent mutant (deficient levels of SLPS; JS1004); lane 2, Km resistance cassette interruption of rsaA (wild-type levels of SLPS; JS1003); lane 3, wild-type C. crescentus, NA1000; lane 4, manB knockout in S-layer-negative strain (no SLPS; JS1014). Presence (+) or absence (−) of manB and S-layer (SL) is indicated. (b) S-layer attachment requires SLPS. Immunoblot analysis using RsaA antiserum. Wild-type RsaA was incubated with S-layer-negative cells possessing various SLPS levels: wild-type (JS1003), ++; deficient (JS1004), +; or none (JS1014), −. Whole-cell protein preparations from equivalent numbers of cells were then performed. The lane marked control (ctl) is input RsaA alone. N/A, not applicable.

Region of RsaA that mediates S-layer anchoring.Since disruption of the putative RsaA anchoring or crystallization domains results in S-layer shedding, when a mutation results in a shedding phenotype, it is difficult to know whether RsaA anchoring or crystallization has been perturbed. The goal, then, was to identify a portion of RsaA that was sufficient for anchoring yet small enough that it would likely be able to do so only by direct interaction with the cell surface, rather than by RsaA subunit-subunit interactions.

To that end, rsaA variants with collagenase cleavage sites at selected positions in RsaA were constructed. Soluble protein was isolated from culture supernatants of shedder strains and treated with collagenase, and the resulting fragments were incubated with S-layer-negative cells. Initial results from several experiments suggested that both the RsaA N- and C-terminal fragments reattached to S-layer-negative cells (not shown). Upon closer inspection, it appeared that the RsaA N-terminal fragment always reattached, while the RsaA C-terminal fragment only reattached when residual full-length (uncleaved) RsaA was available and also reattached (not shown). This suggested that the reattachment of the RsaA C-terminal fragment may be due to an association of C-terminal fragments with residual full-length RsaA rather than to direct attachment of the RsaA C-terminal fragment to the cell surface. To test this hypothesis, RsaA bearing a collagenase cleavage site at residue 277 was treated with collagenase for various lengths of time to generate partial and complete digests of the protein. The digested RsaA preparations were then incubated with S-layer-negative cells (JS4015) to assess attachment. As hypothesized, the RsaA C-terminal fragment (residues 278 to 1026) only reattached when some full-length RsaA was available and also reattached (Fig. 3, compare lane 2 with lane 5), suggesting that the RsaA C-terminal fragment contained no anchoring information. In addition, while smaller RsaA N-terminal fragments could not be readily detected with RsaA antiserum (data not shown), these data also support the conclusion that residues 1 to 277 RsaA of [Rsa(1-277)] is sufficient for RsaA anchoring. Note that RsaA(1-277) appears to run anomalously at ∼34 kDa rather than the expected ∼28 kDa. The weak band at ∼28 k is likely due to a low level of Sap activity still present in JS4015; this point is further discussed below.

FIG. 3.
  • Open in new tab
  • Download powerpoint
FIG. 3.

RsaA C-terminal fragment anchoring requires full-length RsaA. Immunoblot analysis was performed using RsaA antiserum. RsaA bearing a collagenase cleavage site at residue 277 was treated with collagenase for increasing lengths of time. The resulting RsaA fragments were incubated with Sap-negative, S-layer-negative cells (JS4015). After a washing step, preparations from equivalent numbers of cells were examined. Lane 1, JS4015 cells (no treatment, N/T); lanes 2 to 5, JS4015 cells plus RsaA digested by collagenase for various lengths of time (60, 120, or 180 min or overnight); lane 6, RsaA digested by collagenase overnight. O/N, overnight.

Investigation of regions within the RsaA anchoring domain.Initial experiments attempting to reattach full-length RsaA with various N-terminal mutations were unsuccessful (data not shown) and served as preliminary evidence that RsaA anchoring information is located in the RsaA N terminus. To discriminate regions within the RsaA N terminus that are important for anchoring, independent mutations in the N terminus were constructed at positions in RsaA corresponding to amino acids 7, 29, 69, 154, 169, and 222 (Fig. 4a). Mutations at residues 7, 154, and 222 resulted in a two-amino-acid exchange for Gly/Ser, while mutations at residues 29, 69, and 169 resulted in the insertion of four amino acids (N-Asp-Gly-Ser-Val) at these positions. All of these constructs also possessed the collagenase cleavage site at residue 277. Full-length RsaA from the constructs was collected from the C. crescentus shedder strain JS1012 and treated with collagenase. After collagenase treatment, all constructs yielded RsaA fragments of the expected size, as evidenced by immunoblotting (data not shown). The resulting fragments were incubated with Sap-negative (see next section), S-layer-negative cells that were either SLPS positive (JS4015) or negative (JS4024). After washing, the cells were analyzed for the presence of RsaA and RsaA fragments. No RsaA fragments were detected on the SLPS-negative cells, as expected (Fig. 4b). The mutations at RsaA positions 7, 29, and 69 caused loss of anchoring to the SLPS-positive cells (Fig. 4b), as did mutations at positions 154, 169, and 222 (not shown), suggesting that all of the regions of RsaA that were mutated contribute to the anchoring of the RsaA N terminus to SLPS. The digest of RsaA bearing the collagenase site at residue 277, but without further mutation in the N terminus (the control input protein), appeared to go to completion (Fig. 4b, lane ctl). The resulting RsaA(1-277) fragment clearly reattached to SLPS-positive cells, and the C-terminal fragment [RsaA(278-1026)] did not (Fig. 4b, wt lanes). The lack of C-terminal fragment reattachment is consistent with our previous finding that some full-length protein is required for any C-terminal fragment to bind and that only the N terminus of RsaA carries the anchoring information for the protein (Fig. 3). Taken together, these data confirm that the RsaA N terminus mediates RsaA anchoring, and small perturbations within the first ∼225 amino acids disrupt RsaA anchoring.

FIG. 4.
  • Open in new tab
  • Download powerpoint
FIG. 4.

The effect of mutations in the N terminus of RsaA. (a) Linear representation of mutant RsaA used for reattachment studies. Independent mutations at positions in the RsaA N terminus corresponding to amino acids 7, 29, 69, 154, 169, and 222 were constructed. These full-length proteins were isolated from the C. crescentus shedder strain JS1012 and treated with collagenase. The resulting RsaA fragments were assessed for their ability to reattach to S-layer-negative cells. (b) Mutations at RsaA amino acids 7, 29, and 69 disrupt RsaA attachment. After collagenase digestion, RsaA fragments were incubated with Sap-negative cells that either possessed (JS4015; +) or did not possess (JS4024; −) SLPS. After a washing step, whole-cell protein preparations from equivalent numbers of cells were examined by immunoblot analysis using RsaA antiserum. The lane marked control (ctl) is collagenase-treated input RsaA that did not possess an additional mutation in its N terminus. wt, wild type.

Sap localization and proteolytic activity.In initial RsaA reattachment studies, we observed that reattachment of RsaA(1-277) to the cell surface of S-layer-deficient cells also resulted in a single cleavage of RsaA(1-277), such that a shorter product than predicted was noted in the assay. Interestingly, this product of about 28 kDa was the same size as cleavage products previously observed for some other RsaA mutants cleaved by the S-layer-associated protease, Sap (8); the working hypothesis is that this represents a “weak” site that can be cleaved by Sap if RsaA folding results in incomplete or abnormal secondary structure.

To determine if Sap was responsible for the cleavage of RsaA(1-277), soluble RsaA bearing the collagenase site at residue 277 was isolated from either Sap-positive (JS1004) or Sap-negative cells (JS1012). The isolated protein was treated with collagenase, and the resulting RsaA fragments were incubated with S-layer-negative target cells that were either Sap positive (JS1003) or Sap deficient (JS4015). The source of the input protein did not matter in terms of the cleavage of RsaA(1-277), however; the use of Sap-positive target cells led to complete cleavage of this protein, whereas the use of Sap-deficient target cells largely prevented this cleavage (Fig. 5, compare lanes 1 and 4 or lanes 2 and 5). In JS4015, the Sap deficiency derives from a point mutation in the active site (38), which reduces its activity but may not abolish it completely and so may allow for some residual cleavage of RsaA(1-277). This may account for the presence of a small amount of the cleaved version of RsaA(1-277) when JS4015 cells were used as target cells for RsaA(1-277) reattachment (Fig. 5, lanes 1 and 2). The band that is smaller than the cleaved RsaA(1-277) (lanes 4 and 5) and that does not appear in lanes 1 and 2 may result from a poorly recognized cleavage site that is only exposed after an initial cut of RsaA(1-277) by Sap.

FIG. 5.
  • Open in new tab
  • Download powerpoint
FIG. 5.

Sap cleaves RsaA(1-277). RsaA bearing a collagenase site at 277 was isolated from a Sap− (JS1012) or Sap+ (JS1004) shedder (S-layer secreting) strain and then treated with collagenase. The resulting RsaA fragments were then incubated with corresponding numbers of S-layer-negative target cells that are Sap− (JS4015) or Sap+ (JS1003). After a washing step, whole-cell preparations were examined by immunoblot analysis using RsaA antiserum.

These data suggest that Sap harbored by the target cells was responsible for the cleavage of RsaA(1-277). This was a fortuitous result, since the S-layer reattachment assay involved mutating sites within RsaA(1-277) and then testing these fragments for their ability to reattach to S-layer-negative cells. We wanted to be sure that when a particular RsaA mutation led to loss of RsaA anchoring, this loss of anchoring resulted from the fact that the sequence that was mutated contributed directly to anchoring, either by direct attachment of this sequence to SLPS or by contributing to the RsaA structure required for anchoring, rather than simply exposing a previously unrecognized cleavage site. To that end, in the reattachment assays involving mutant RsaA (described above), S-layer-negative strains that are also Sap negative (e.g., JS4015 and JS4024) were used as targets for reattachment (Fig. 4b).

Since the supernatant of the target cell culture was removed prior to exogenous addition of RsaA(1-277) and since RsaA(1-277) is not likely to be imported into the cell, for Sap to be able to cleave RsaA(1-277), it must either be located on the cell surface or released from the cytoplasm in some way. To rule out the possibility that the lysis of some reattachment target cells released Sap from the cytoplasm, allowing it to encounter and cleave RsaA(1-277), some reattachment mixtures were boiled immediately after reattachment of RsaA(1-277), with the expectation that boiling would denature any protease that was hypothetically released from lysed cells. Samples prepared in this way still exhibited the proteolysis of RsaA(1-277) (data not shown), suggesting that the Sap-mediated proteolysis occurred during the reattachment process rather than some time after reattachment due to some cell lysis. These results suggest that Sap is located on the cell surface.

Secretion of Sap to the cell surface.Given the indication that Sap was surface localized, focus shifted to assessing how the protease was secreted. There is no significant degree of homology between the C-terminal secretion signal of RsaA and the C terminus of Sap. However, AprA, a protease from P. aeruginosa whose C terminus shares even less homology to the C terminus of RsaA, can be secreted by the rsaA type I transporter (1); we considered it possible that Sap might also be secreted by this system.

Accordingly, an NA1000-derived strain was constructed that was null for rsaF a and rsaF b, two transporter genes required for the C. crescentus S-layer type I secretion system (37) and for rsaA (JS1011) (Fig. 6a, lane 3); a JS4000-derived strain that was null for rsaF a and rsaF b (JS4025) (Fig. 6a, lane 6) was also constructed. These strains were used as targets for reattachment of RsaA(1-277) to see if Sap proteolytic activity was affected. Cleavage of RsaA(1-277) was significantly reduced when JS1011 was used compared results with a target (UJ2602) that possesses the wild-type outer membrane proteins (OMPs) of the S-layer type I transporter proteins (Fig. 6b, compare lanes 3 and 4). This suggests that RsaFa and/or RsaFb is involved in the secretion of Sap in strain NA1000, and thus when the OMPs are not present, Sap largely does not get secreted and therefore cannot access and cleave RsaA(1-277) that has reattached to the cell surface.

FIG. 6.
  • Open in new tab
  • Download powerpoint
FIG. 6.

The effect of RsaFa and RsaFb levels on Sap secretion. (a) RsaFa and RsaFb levels in wild-type and RsaF knockout strains. Whole-cell protein preparations from equivalent numbers of NA1000 (wt) or JS4000 (wt) cells or strains derived from them that are null for RsaFa (Fa−; JS4023), RsaFb (Fb −; JS1008), or both RsaFa and RsaFb (Fa −/Fb −; JS1011 and JS4025) were examined by immunoblot analysis using RsaF antiserum. (b) S-layer type I secretion OMP RsaFa and/or RsaFb is involved in the secretion of Sap. RsaA bearing a collagenase cleavage site at residue 277 was isolated from the Sap− C. crescentus shedder strain JS1012 (lane 2) and then treated with collagenase (lane 1). The resulting RsaA fragments were then incubated with corresponding numbers of S-layer-negative C. crescentus cells derived from NA1000 that either possess (UJ2602, lane 4) or do not possess (JS1011, lane 3) RsaFa and RsaFb. After washing, cells were immediately boiled in the presence of SDS-PAGE loading buffer. Equal loadings were examined by immunoblot analysis using RsaA antiserum. (c) A third RsaF OMP may be present in strain JS4025. RsaA bearing a collagenase cleavage site at residue 277 was isolated from the Sap− C. crescentus shedder strain JS1012 and then treated with collagenase (lane 4). The resulting RsaA fragments were then incubated with corresponding numbers of cells from JS1011 (lane 1) or JS4025 (lane 2). As a control, the RsaA fragments were also incubated with JS4015 cells (lane 3). After washing, cells were immediately boiled in the presence of loading buffer. Equal loadings were examined by immunoblot analysis using RsaA antiserum.

Strain JS4000 is a strain of distinct lineage from NA1000, though it is capable of producing an RsaA that is identical to that of NA1000, except for an apparent spontaneous frameshift mutation in rsaA that results in an early stop codon (33). A search of the NA1000 genome (www.tigr.org ) suggested that there are only two chromosomal OMP genes (rsaF a and rsaF b) involved in S-layer type I secretion, and this was confirmed experimentally (37). In contrast to JS1011 (derived from NA1000), upon introduction of a plasmid-borne copy of rsaA into the JS4000-derived JS4025, RsaA was still secreted (not shown). This suggested that a third rsaF gene may be present on the JS4000 chromosome, allowing for RsaA secretion when rsaF a and rsaF b have been inactivated. To gather more evidence of a third RsaF OMP in JS4000 and to demonstrate that Sap can utilize this putative OMP for secretion, cleavage of RsaA(1-277) was assessed using JS4025 as a target for reattachment of RsaA(1-277). Full cleavage of RsaA(1-277) was observed when JS4025 (Fig. 6c, lane 2) but not JS1011 (Fig. 6c, lane 1) was used as a target. Taken together, these data suggest that a third RsaF protein is present in JS4000, and this third RsaF transporter protein is also involved in the secretion of Sap.

DISCUSSION

We developed an assay that can be used to systematically investigate regions or residues of the C. crescentus S-layer protein RsaA that are engaged in S-layer anchoring. In the course of assay development, we developed and improved a method of obtaining RsaA that significantly improved functional activity. We demonstrated that the O side chain of SLPS was required for RsaA anchoring and established that the N terminus of RsaA mediates anchoring of the S-layer to the cell surface, and we learned that the first 277 amino acids are sufficient for RsaA anchoring. Surprisingly, all six mutations created within the first 222 amino acids resulted in loss of RsaA anchoring, suggesting that this entire region contributes to S-layer anchoring. It became necessary for us to better understand the activity and localization of the S-layer-associated protease, Sap, such that data gathered from reattachment experiments reflected actual S-layer anchoring rather than Sap proteolytic activity. In doing so, we accumulated evidence that Sap is likely localized on the cell surface rather than in the cytoplasm as previously proposed, which implicated the S-layer type I secretion OMP RsaFa and/or RsaFb in Sap secretion to the cell surface.

Although the SLH motif is well documented for gram-positive bacteria (16), there is no such motif defined for S-layer proteins of gram-negative bacteria. Indeed, little work has been done to define S-layer anchoring regions in gram-negative bacteria. To our knowledge, there has only been one other study that evaluated the ability of a truncated S-layer protein to reattach to S-layer-deficient gram-negative cells. In that study, deletion mutagenesis revealed that C. fetus S-layer proteins bound serospecifically to the C. fetus lipopolysaccharide via conserved N-terminal regions, which include approximately 189 amino acids (14). Those findings are comparable to the results of this study for the C. crescentus S-layer protein: the first ∼225 amino acids of RsaA are involved in S-layer anchoring, suggesting that a large structural region may be essential to mediate S-layer anchoring in gram-negative bacteria. This contrasts with anchoring domains for S-layer proteins of gram-positive bacteria, which are usually three repeats of the ∼55-amino-acid SLH domain (4).

Although the native function of Sap is unknown, our previous model of Sap-mediated editing of the recombinant S-layer suggested that because the C terminus of Sap is homologous with the N terminus of RsaA, Sap associates with nascent, but still internal, RsaA monomers and cleaves some mutant versions of RsaA (38). A recombinant S-layer protein subjected to Sap proteolysis would result in the separation of the RsaA C-terminal secretion signal from the rest of RsaA. Confusingly, however, both Sap cleavage products usually appeared on the cell surface, though only one of these products contains the C-terminal secretion signal to effect secretion to the cell surface (38). Since we had previously predicted that Sap was not on the cell surface (an analysis of the Sap sequence reveals no predicted N-terminal signal leader peptide, no predicted type I secretion signal, and no predicted transmembrane domains), we hypothesized that after Sap-mediated proteolysis in the cytoplasm, intramolecular forces (such as hydrogen bonding) kept the two Sap cleavage products linked together, accounting for their simultaneous secretion.

However, evidence here suggests that Sap is located on the cell surface and can access and cleave reattached RsaA(1-277). If so, some Sap substrates [such as RsaA:VP2CΔ (38) and RsaA(1-277)] would be cleaved by Sap on the cell surface rather than in the cytoplasm. Thus far, efforts to examine the culture supernatant have concluded that little or no Sap is found free of cells. Clearly, additional confirmations of the localization of Sap using tools such as Sap-specific antibody are the next steps.

We have observed that Sap frequently cleaves at the site of a heterologous insertion but then also cleaves various recombinant RsaA mutants at a second site that in each case is the same position in RsaA, yielding a ∼28-kDa RsaA N-terminal cleavage product (see reference 7). This apparent weak site is present in wild-type RsaA, but Sap does not cleave wild-type RsaA. We hypothesize that this Sap recognition site is normally inaccessible to Sap due to appropriate folding of the S-layer. In contrast, mutations in RsaA may cause altered or slower folding of the S-layer, thus exposing this weak cleavage site to Sap. Thus, with a relatively simple mechanism, monomers containing errors anywhere along their lengths that affect the assembly function of S-layer can be cleaved and released after secretion and so not disrupt assembly of the normal protein. It is also possible that the protease could be part of a “remove and replace” strategy for repairing S-layer damaged by various environmental causes. In the current experiments, this site is apparently also exposed when N-terminally truncated proteins are applied. Efforts are under way to determine the exact position and recognition sequence of this Sap cleavage site.

The involvement of RsaFa and/or RsaFb in Sap secretion indicates that Sap secretion is aided or accomplished by the S-layer type I transporter. Nevertheless, only partial loss of activity in an rsaF a/rsaF b null mutant is perplexing. The simplest possibility is that another OMP-like protein, such as one involved in drug export, in concert with the remainder of the S-layer transporter may enable some Sap secretion. But other explanations, such as the type I secretion of another factor that positively influences Sap activity, cannot yet be ruled out.

The results also predict the presence of a third OMP in JS4000-based strains that is involved in Sap (and RsaA) secretion. We have noted that JS4000-derived strains frequently produce more recombinant RsaA-derived proteins than the C. crescentus CB15 counterparts; this third OMP may be responsible for this informal observation.

The Sap secretion results may also reinforce the idea that examination of the C-terminal primary sequence of proteins cannot be used to accurately predict or rule out a type I secretion signal. In addition, assuming that the S-layer type I transporter also secretes Sap, this would not be the first type I secretion system shown to secrete multiple proteins. For example, the LipBCD type I secretion system in S. marcescens secretes three proteins: a lipase, a metalloprotease, and the S. marcescens S-layer protein (23).

Experimental evidence suggested that Sap is bound to the outer membrane. If so, after translocation, Sap must tether to molecules on the cell surface. A portion of the C terminus of Sap is homologous to a region in the N terminus of RsaA, and there may be an interaction that mimics the crystallization process. If so, the crystalline RsaA may mediate anchoring of Sap (while the S-layer, in turn, is anchored via SLPS). A plausible alternative is that the Sap C-terminal homology to the N terminus of RsaA reflects a common theme for attachment; thus, the C terminus of Sap anchors to SLPS on the cell surface in the same way that the N terminus of RsaA anchors to SLPS. Efforts are under way to resolve these possibilities.

The type V autotransporter secretion (21) and lipid-linked proteins (3) are two examples in which proteins are secreted and subsequently anchored to the cell envelope of gram-negative bacteria. Both of these transport systems utilize the Sec-dependent general secretory pathway to traverse the inner membrane and after traversing the periplasm subsequently enter the outer membrane. In contrast to the general secretory pathway, the type I transporter secretes proteins across both the inner and outer membranes without generating periplasmic intermediates (28). If it is true that the C. crescentus S-layer type I secretion system also secretes other proteins such as Sap which subsequently anchor to SLPS on the cell surface, then we may have discovered still another process involved in the secretion and anchoring of proteins to the cell surface that could extend to other gram-negative bacteria.

ACKNOWLEDGMENTS

This work was supported by grants from the Canadian Natural Sciences and Engineering Research Council to J.S. and by a University Graduate Fellowship from the University of British Columbia to M.J.F.

FOOTNOTES

    • Received 1 November 2006.
    • Accepted 21 December 2006.
  • Copyright © 2007 American Society for Microbiology

REFERENCES

  1. 1.↵
    Awram, P., and J. Smit. 1998. The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus. J. Bacteriol. 180 : 3062-3069.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    Awram, P., and J. Smit. 2001. Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus. Microbiology 147 : 1451-1460.
    OpenUrlCrossRefPubMed
  3. 3.↵
    Bernstein, H. D. 2000. The biogenesis and assembly of bacterial membrane proteins. Curr. Opin. Microbiol. 3 : 203-209.
    OpenUrlCrossRefPubMed
  4. 4.↵
    Beveridge, T. J., and L. L. Graham. 1991. Surface layers of bacteria. Microbiol. Rev. 55 : 684-705.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Beveridge, T. J., P. H. Pouwels, M. Sara, A. Kotiranta, K. Lounatmaa, K. Kari, E. Kerosuo, M. Haapasalo, E. M. Egelseer, I. Schocher, U. B. Sleytr, L. Morelli, M. L. Callegari, J. F. Nomellini, W. H. Bingle, J. Smit, E. Leibovitz, M. Lemaire, I. Miras, S. Salamitou, P. Beguin, H. Ohayon, P. Gounon, M. Matuschek, and S. F. Koval. 1997. Functions of S-layers. FEMS Microbiol. Rev. 20 : 99-149.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    Binet, R., S. Letoffe, J. M. Ghigo, P. Delepelaire, and C. Wandersman. 1997. Protein secretion by gram-negative bacterial ABC exporters—a review. Gene 192 : 7-11.
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    Bingle, W. H., J. F. Nomellini, and J. Smit. 1997. Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus. Mol. Microbiol. 26 : 277-288.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    Bingle, W. H., J. F. Nomellini, and J. Smit. 1997. Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion. J. Bacteriol. 179 : 601-611.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    Bingle, W. H., J. F. Nomellini, and J. Smit. 2000. Secretion of the Caulobacter crescentus S-layer protein: further localization of the C-terminal secretion signal and its use for secretion of recombinant proteins. J. Bacteriol. 182 : 3298-3301.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Bingle, W. H., and J. Smit. 1990. High-level expression vectors for Caulobacter crescentus incorporating the transcription/translation initiation regions of the paracrystalline surface-layer-protein gene. Plasmid 24 : 143-148.
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    Boot, H. J., and P. H. Pouwels. 1996. Expression, secretion and antigenic variation of bacterial S-layer proteins. Mol. Microbiol. 21 : 1117-1123.
    OpenUrlCrossRefPubMed
  12. 12.
    Brent, R., and M. Ptashne. 1980. The lexA gene product represses its own promoter. Proc. Natl. Acad. Sci. USA 77 : 1932-1936.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    Doig, P., W. D. McCubbin, C. M. Kay, and T. J. Trust. 1993. Distribution of surface-exposed and non-accessible amino acid sequences among the two major structural domains of the S-layer protein of Aeromonas salmonicida. J. Mol. Biol. 233 : 753-765.
    OpenUrlCrossRefPubMed
  14. 14.↵
    Dworkin, J., M. K. Tummuru, and M. J. Blaser. 1995. A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs. J. Bacteriol. 177 : 1734-1741.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Edwards, P., and J. Smit. 1991. A transducing bacteriophage for Caulobacter crescentus uses the paracrystalline surface layer protein as a receptor. J. Bacteriol. 173 : 5568-5572.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    Engelhardt, H., and J. Peters. 1998. Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer-cell wall interactions. J. Struct. Biol. 124 : 276-302.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.
    Evinger, M., and N. Agabian. 1977. Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J. Bacteriol. 132 : 294-301.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Garduno, R. A., B. M. Phipps, and W. W. Kay. 1995. Physical and functional S-layer reconstitution in Aeromonas salmonicida. J. Bacteriol. 177 : 2684-2694.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Gilchrist, A., J. A. Fisher, and J. Smit. 1992. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can. J. Microbiol. 38 : 193-202.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Gilchrist, A., and J. Smit. 1991. Transformation of freshwater and marine caulobacters by electroporation. J. Bacteriol. 173 : 921-925.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Henderson, I. R., F. Navarro-Garcia, M. Desvaux, R. C. Fernandez, and D. Ala'Aldeen. 2004. Type V protein secretion pathway: the autotransporter story. Microbiol. Mol. Biol. Rev. 68 : 692-744.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Iuga, M., P. Awram, J. F. Nomellini, and J. Smit. 2004. Comparison of S-layer secretion genes in freshwater caulobacters. Can. J. Microbiol. 50 : 751-766.
    OpenUrlCrossRefPubMed
  23. 23.↵
    Kawai, E., H. Akatsuka, A. Idei, T. Shibatani, and K. Omori. 1998. Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system. Mol. Microbiol. 27 : 941-952.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Lupas, A., H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister. 1994. Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis. J. Bacteriol. 176 : 1224-1233.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    Messing, J., and J. Vieira. 1982. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19 : 269-276.
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    Morales, V. M., A. Backman, and M. Bagdasarian. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97 : 39-47.
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    Nomellini, J. F., S. Kupcu, U. B. Sleytr, and J. Smit. 1997. Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer. J. Bacteriol. 179 : 6349-6354.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    Salmond, G. P., and P. J. Reeves. 1993. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem. Sci. 18 : 7-12.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  30. 30.↵
    Sara, M., and U. B. Sleytr. 2000. S-Layer proteins. J. Bacteriol. 182 : 859-868.
    OpenUrlFREE Full Text
  31. 31.↵
    Schäfer, A., A. Tauch, W. Jäger, J. Kalinowski, and G. Theirbach. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Cornyebacterium glutamicum. Gene 145 : 69-73.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    Sleytr, U. B., and T. J. Beveridge. 1999. Bacterial S-layers. Trends Microbiol. 7 : 253-260.
    OpenUrlCrossRefPubMedWeb of Science
  33. 33.↵
    Smit, J., and N. Agabian. 1984. Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays. J. Bacteriol. 160 : 1137-1145.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    Smit, J., H. Engelhardt, S. Volker, S. H. Smith, and W. Baumeister. 1992. The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy. J. Bacteriol. 174 : 6527-6538.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    Thompson, S. A., O. L. Shedd, K. C. Ray, M. H. Beins, J. P. Jorgensen, and M. J. Blaser. 1998. Campylobacter fetus surface layer proteins are transported by a type I secretion system. J. Bacteriol. 180 : 6450-6458.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    Toporowski, M. C., J. F. Nomellini, P. Awram, A. Levi, and J. Smit. 2005. Transcriptional regulation of the S-layer protein type I secretion system in Caulobacter crescentus. FEMS Microbiol. Lett. 251 : 29-36.
    OpenUrlCrossRefPubMed
  37. 37.↵
    Toporowski, M. C., J. F. Nomellini, P. Awram, and J. Smit. 2004. Two outer membrane proteins are required for maximal type I secretion of the Caulobacter crescentus S-layer protein. J. Bacteriol. 186 : 8000-8009.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    Umelo-Njaka, E., W. H. Bingle, F. Borchani, K. D. Le, P. Awram, T. Blake, J. F. Nomellini, and J. Smit. 2002. Caulobacter crescentus synthesizes an S-layer-editing metalloprotease possessing a domain sharing sequence similarity with its paracrystalline S-layer protein. J. Bacteriol. 184 : 2709-2718.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    Umelo-Njaka, E., J. F. Nomellini, H. Yim, and J. Smit. 2001. Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus. Plasmid 46 : 37-46.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Walker, S. G., D. N. Karunaratne, N. Ravenscroft, and J. Smit. 1994. Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer. J. Bacteriol. 176 : 6312-6323.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    Walker, S. G., S. H. Smith, and J. Smit. 1992. Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters. J. Bacteriol. 174 : 1783-1792.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    Welch, R. A., C. Forestier, A. Lobo, S. Pellett, W. Thomas, Jr., and G. Rowe. 1992. The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins. FEMS Microbiol. Immunol. 5 : 29-36.
    OpenUrlCrossRefPubMed
  43. 43.↵
    Yang, L. Y., Z. H. Pei, S. Fujimoto, and M. J. Blaser. 1992. Reattachment of surface array proteins to Campylobacter fetus cells. J. Bacteriol. 174 : 1258-1267.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus
Matthew J. Ford, John F. Nomellini, John Smit
Journal of Bacteriology Feb 2007, 189 (6) 2226-2237; DOI: 10.1128/JB.01690-06

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Bacteriology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus
(Your Name) has forwarded a page to you from Journal of Bacteriology
(Your Name) thought you would be interested in this article in Journal of Bacteriology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus
Matthew J. Ford, John F. Nomellini, John Smit
Journal of Bacteriology Feb 2007, 189 (6) 2226-2237; DOI: 10.1128/JB.01690-06
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Bacterial Proteins
Caulobacter crescentus
Cell Membrane
Endopeptidases
Membrane Glycoproteins
Protein Binding

Related Articles

Cited By...

About

  • About JB
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jbacteriology

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0021-9193; Online ISSN: 1098-5530